Project Summary/Abstract Coccidioides immitis and Coccidioides posadasii are the fungal species responsible for the disease termed coccidioidomycosis or Valley fever, which is designated as an orphan disease by the FDA. Coccidioides species are endemic in the soil throughout California, Arizona, Mexico, and Central and South America. On average, Valley fever infects an estimated 150,000 individuals per year in the United States, with cases steadily rising as climate temperatures increase in endemic areas. There is no definitive diagnostic available for Valley fever. Currently, patients are diagnosed by chest x- rays that rely heavily on the experience of clinicians, sputum culture microbiological examination/enumeration, and blood sample tests for antibodies. These diagnostic methods suffer from low sensitivity and slow turnaround times (days to often weeks), and are typically made after the patient has displayed severe symptoms of infection. Therefore, an urgent need exists to develop a rapid and non-invasive method for detecting Coccidioides. In addition, this diagnostic should be sufficiently sensitive to enable early detection of the pathogen, prior to the onset of severe or life-threatening symptoms. The overall objective of this R21 proposal is to begin the exploratory pathway for a rapid, sensitive, and non-invasive method for detecting Coccidioides that would significantly change current clinical practice by enabling the early identification of Valley fever, before it has had the chance to become life-threatening. Our proposal focuses on using Coccidioides-specific proteases as a target for development of a diagnostic. To do this, in Aim 1, we use a technology called multiplex substrate profiling by mass spectrometry (MSP-MS) to detect proteolytic activity in cell-free secretions from C. immitis and C. posadasii and use this information to develop protease-cleavable fluorogenic substrates. MSP-MS allows for unbiased and simultaneous detection of all protease activities in a given sample; it employs a library of rationally designed peptide substrates and monitors their cleavage. In Aim 2 we evaluate our fluorogenic substrates in preclinical mouse models of coccidioidomycosis to provide feasibility for downstream human sample testing. If successful, this R21 will set the framework for the development of a substrate-based diagnostic for the detection of Coccidioides in Valley fever patients.